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1.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445316

RESUMO

Inhibition of pyruvate dehydrogenase kinase (PDK) emerged as a potential strategy for treatment of cancer and metabolic disorders. Dichloroacetate (DCA), a prototypical PDK inhibitor, reduces the abundance of some PDK isoenzymes. However, the underlying mechanisms are not fully characterized and may differ across cell types. We determined that DCA reduced the abundance of PDK1 in breast (MDA-MB-231) and prostate (PC-3) cancer cells, while it suppressed both PDK1 and PDK2 in skeletal muscle cells (L6 myotubes). The DCA-induced PDK1 suppression was partially dependent on hypoxia-inducible factor-1α (HIF-1α), a transcriptional regulator of PDK1, in cancer cells but not in L6 myotubes. However, the DCA-induced alterations in the mRNA and the protein levels of PDK1 and/or PDK2 did not always occur in parallel, implicating a role for post-transcriptional mechanisms. DCA did not inhibit the mTOR signaling, while inhibitors of the proteasome or gene silencing of mitochondrial proteases CLPP and AFG3L2 did not prevent the DCA-induced reduction of the PDK1 protein levels. Collectively, our results suggest that DCA reduces the abundance of PDK in an isoform-dependent manner via transcriptional and post-transcriptional mechanisms. Differential response of PDK isoenzymes to DCA might be important for its pharmacological effects in different types of cells.


Assuntos
Ácido Dicloroacético/farmacologia , Inibidores Enzimáticos/farmacologia , Fibras Musculares Esqueléticas/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil/antagonistas & inibidores , Proteases Dependentes de ATP/antagonistas & inibidores , Proteases Dependentes de ATP/metabolismo , ATPases Associadas a Diversas Atividades Celulares/antagonistas & inibidores , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Linhagem Celular Tumoral , Endopeptidase Clp/antagonistas & inibidores , Endopeptidase Clp/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Células PC-3 , Piruvato Desidrogenase Quinase de Transferência de Acetil/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , Ratos
2.
Commun Biol ; 4(1): 841, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230602

RESUMO

Characterizing protein-protein interactions (PPIs) is an effective method to help explore protein function. Here, through integrating a newly identified split human Rhinovirus 3 C (HRV 3 C) protease, super-folder GFP (sfGFP), and ClpXP-SsrA protein degradation machinery, we developed a fluorescence-assisted single-cell methodology (split protease-E. coli ClpXP (SPEC)) to explore protein-protein interactions for both eukaryotic and prokaryotic species in E. coli cells. We firstly identified a highly efficient split HRV 3 C protease with high re-assembly ability and then incorporated it into the SPEC method. The SPEC method could convert the cellular protein-protein interaction to quantitative fluorescence signals through a split HRV 3 C protease-mediated proteolytic reaction with high efficiency and broad temperature adaptability. Using SPEC method, we explored the interactions among effectors of representative type I-E and I-F CRISPR/Cas complexes, which combining with subsequent studies of Cas3 mutations conferred further understanding of the functions and structures of CRISPR/Cas complexes.


Assuntos
Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Mapas de Interação de Proteínas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas CRISPR-Cas , Endopeptidase Clp/genética , Enterovirus/enzimologia , Enterovirus/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histidina Quinase/genética , Histidina Quinase/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteólise , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
3.
Int J Mol Sci ; 22(12)2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207660

RESUMO

Caseinolytic protease P (ClpP) is a mitochondrial serine protease. In mammalian cells, the heterodimerization of ClpP and its AAA+ ClpX chaperone results in a complex called ClpXP, which has a relevant role in protein homeostasis and in maintaining mitochondrial functionality through the degradation of mitochondrial misfolded or damaged proteins. Recent studies demonstrate that ClpP is upregulated in primary and metastatic human tumors, supports tumor cell proliferation, and its overexpression desensitizes cells to cisplatin. Interestingly, small modulators of ClpP activity, both activators and inhibitors, are able to impair oxidative phosphorylation in cancer cells and to induce apoptosis. This review provides an overview of the role of ClpP in regulating mitochondrial functionality, in supporting tumor cell proliferation and cisplatin resistance; finally, we discuss whether this protease could represent a new prognostic marker and therapeutic target for the treatment of cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Endopeptidase Clp/metabolismo , Mitocôndrias/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/terapia , Animais , Apoptose/genética , Biomarcadores Tumorais/genética , Proliferação de Células/genética , Endopeptidase Clp/genética , Humanos , Mitocôndrias/genética , Mitocôndrias/patologia , Metástase Neoplásica , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia
4.
Protein Sci ; 30(4): 899-907, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33599047

RESUMO

In the model organism Escherichia coli and related species, the general stress response relies on tight regulation of the intracellular levels of the promoter specificity subunit RpoS. RpoS turnover is exclusively dependent on RssB, a two-domain response regulator that functions as an adaptor that delivers RpoS to ClpXP for proteolysis. Here, we report crystal structures of the receiver domain of RssB both in its unphosphorylated form and bound to the phosphomimic BeF3 - . Surprisingly, we find only modest differences between these two structures, suggesting that truncating RssB may partially activate the receiver domain to a "meta-active" state. Our structural and sequence analysis points to RssB proteins not conforming to either the Y-T coupling scheme for signaling seen in prototypical response regulators, such as CheY, or to the signaling model of the less understood FATGUY proteins.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Transdução de Sinais , Fatores de Transcrição/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endopeptidase Clp/química , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fator sigma/química , Fator sigma/genética , Fator sigma/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
5.
J Biol Chem ; 296: 100460, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33639171

RESUMO

Bacterial survival during lethal heat stress relies on the cellular ability to reactivate aggregated proteins. This activity is typically executed by the canonical 70-kDa heat shock protein (Hsp70)-ClpB bichaperone disaggregase, which is most widespread in bacteria. The ClpB disaggregase is a member of the ATPase associated with diverse cellular activities protein family and exhibits an ATP-driven threading activity. Substrate binding and stimulation of ATP hydrolysis depends on the Hsp70 partner, which initiates the disaggregation reaction. Recently elevated heat resistance in gamma-proteobacterial species was shown to be mediated by the ATPase associated with diverse cellular activities protein ClpG as an alternative disaggregase. Pseudomonas aeruginosa ClpG functions autonomously and does not cooperate with Hsp70 for substrate binding, enhanced ATPase activity, and disaggregation. With the underlying molecular basis largely unknown, the fundamental differences in ClpG- and ClpB-dependent disaggregation are reflected by the presence of sequence alterations and additional ClpG-specific domains. By analyzing the effects of mutants lacking ClpG-specific domains and harboring mutations in conserved motifs implicated in ATP hydrolysis and substrate threading, we show that the N-terminal, ClpG-specific N1 domain generally mediates protein aggregate binding as the molecular basis of autonomous disaggregation activity. Peptide substrate binding strongly stimulates ClpG ATPase activity by overriding repression by the N-terminal N1 and N2 domains. High ATPase activity requires two functional nucleotide binding domains and drives substrate threading which ultimately extracts polypeptides from the aggregate. ClpG ATPase and disaggregation activity is thereby directly controlled by substrate availability.


Assuntos
Antígenos de Bactérias/metabolismo , Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Antígenos de Bactérias/fisiologia , Endopeptidase Clp/fisiologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/fisiologia , Agregados Proteicos , Ligação Proteica , Domínios Proteicos/genética
6.
Res Microbiol ; 172(2): 103797, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33460738

RESUMO

Antibiotic acyldepsipeptide (ADEP) targets the bacterial ClpP serine protease and can inhibit the growth of numerous bacterial species by activating/dysregulating the protease activity within the cell. The spirochete Leptospira interrogans harbors two ClpP isoforms (LepClpP1 and LepClpP2). Supplementation of ADEP in the Leptospira growth medium resulted in the inhibition of bacterial growth. The ADEP mediated activation of the LepClpP mixture was dependent on the time allowed for the self-assembly of LepClpP1 and LepClpP2. The dynamic light scattering of the LepClpP mixture in the presence of the ADEP indicated a conformational transformation of the LepClpP machinery. Serine 98, a catalytic triad residue of the LepClpP1 in the LepClpP1P2 heterocomplex, was critical for the ADEP mediated activation. The computational prototype of the LepClpP1P2 structure suggested that the hydrophobic pockets wherein the ADEPs or the physiological chaperone ClpX predominantly dock are exclusively present in the LepClpP2 heptamer. Using the ADEP as a tool, this investigation provides an insight into the molecular function of the LepClpP1P2 in a coalition with its ATPase chaperone LepClpX. The shreds of the evidence illustrated in this investigation verify that ADEP1 possesses the ability to control the LepClpP system in an unconventional approach than the other organisms.


Assuntos
Antibacterianos/farmacologia , Depsipeptídeos/farmacologia , Endopeptidase Clp/metabolismo , Leptospira/efeitos dos fármacos , Leptospira/enzimologia , Proteólise/efeitos dos fármacos , Endopeptidase Clp/genética , Interações Hidrofóbicas e Hidrofílicas , Microscopia Eletrônica de Varredura , Chaperonas Moleculares/metabolismo , Simulação de Acoplamento Molecular , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
7.
J Biol Chem ; 296: 100338, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33497624

RESUMO

ATPases Associated with diverse cellular Activities (AAA+) are a superfamily of proteins that typically assemble into hexameric rings. These proteins contain AAA+ domains with two canonical motifs (Walker A and B) that bind and hydrolyze ATP, allowing them to perform a wide variety of different functions. For example, AAA+ proteins play a prominent role in cellular proteostasis by controlling biogenesis, folding, trafficking, and degradation of proteins present within the cell. Several central proteolytic systems (e.g., Clp, Deg, FtsH, Lon, 26S proteasome) use AAA+ domains or AAA+ proteins to unfold protein substrates (using energy from ATP hydrolysis) to make them accessible for degradation. This allows AAA+ protease systems to degrade aggregates and large proteins, as well as smaller proteins, and feed them as linearized molecules into a protease chamber. This review provides an up-to-date and a comparative overview of the essential Clp AAA+ protease systems in Cyanobacteria (e.g., Synechocystis spp), plastids of photosynthetic eukaryotes (e.g., Arabidopsis, Chlamydomonas), and apicoplasts in the nonphotosynthetic apicomplexan pathogen Plasmodium falciparum. Recent progress and breakthroughs in identifying Clp protease structures, substrates, substrate adaptors (e.g., NblA/B, ClpS, ClpF), and degrons are highlighted. We comment on the physiological importance of Clp activity, including plastid biogenesis, proteostasis, the chloroplast Protein Unfolding Response, and metabolism, across these diverse lineages. Outstanding questions as well as research opportunities and priorities to better understand the essential role of Clp systems in cellular proteostasis are discussed.


Assuntos
Apicoplastos/enzimologia , Cianobactérias/enzimologia , Endopeptidase Clp/metabolismo , Plastídeos/enzimologia , Endopeptidase Clp/química , Plasmodium falciparum/enzimologia , Proteômica , Proteostase , Transdução de Sinais , Especificidade por Substrato
8.
Nat Commun ; 12(1): 281, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436616

RESUMO

A functional association is uncovered between the ribosome-associated trigger factor (TF) chaperone and the ClpXP degradation complex. Bioinformatic analyses demonstrate conservation of the close proximity of tig, the gene coding for TF, and genes coding for ClpXP, suggesting a functional interaction. The effect of TF on ClpXP-dependent degradation varies based on the nature of substrate. While degradation of some substrates are slowed down or are unaffected by TF, surprisingly, TF increases the degradation rate of a third class of substrates. These include λ phage replication protein λO, master regulator of stationary phase RpoS, and SsrA-tagged proteins. Globally, TF acts to enhance the degradation of about 2% of newly synthesized proteins. TF is found to interact through multiple sites with ClpX in a highly dynamic fashion to promote protein degradation. This chaperone-protease cooperation constitutes a unique and likely ancestral aspect of cellular protein homeostasis in which TF acts as an adaptor for ClpXP.


Assuntos
Endopeptidase Clp/metabolismo , Chaperonas Moleculares/metabolismo , Proteólise , Sítios de Ligação , Endopeptidase Clp/química , Escherichia coli/genética , Proteínas de Escherichia coli , Deleção de Genes , Genoma Bacteriano , Espectroscopia de Ressonância Magnética , Modelos Biológicos , Modelos Moleculares , Mutagênese , Peptídeos/metabolismo , Peptidilprolil Isomerase , Filogenia , Ligação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , Multimerização Proteica , Ribossomos/metabolismo , Especificidade por Substrato , Proteínas Virais/metabolismo
9.
Int J Biol Macromol ; 171: 414-422, 2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33428953

RESUMO

Protein arginine phosphorylation (pArg), a novel molecular switch, plays a key role in regulating cellular processes. The intrinsic acid lability, hot sensitivity, and hot-alkali instability of "high-energy" phosphoamidate (PN bond) in pArg, make the investigation highly difficult and challenging. Recently, the progress in identifying prokaryotic protein arginine kinase/phosphatase and assigning hundreds of pArg proteins and phosphosites has been made, which is arousing scientists' interest and passions. It shows that pArg is tightly connected to bacteria stress response and pathogenicity, and is probably implied in human diseases. In this review, we highlight the strategies for investigation of this mysterious modification and its momentous physiological functions, and also prospect for the potentiality of drugs development targeting pArg-relative pathways.


Assuntos
Arginina/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Bactérias/metabolismo , Endopeptidase Clp/metabolismo , Proteínas de Choque Térmico/imunologia , Proteínas de Choque Térmico/metabolismo , Humanos , Espectrometria de Massas , Fosfatos/metabolismo , Fosfoproteínas/imunologia , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Estresse Fisiológico , Transcrição Genética , Virulência
10.
J Mol Biol ; 433(5): 166815, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33450247

RESUMO

Bacterial toxin-antitoxin (TA) systems are composed of a deleterious toxin and its antagonistic antitoxin. They are widespread in bacterial genomes and mobile genetic elements, and their functions remain largely unknown. Some TA systems, known as TAC modules, include a cognate SecB-like chaperone that assists the antitoxin in toxin inhibition. Here, we have investigated the involvement of proteases in the activation cycle of the TAC system of the human pathogen Mycobacterium tuberculosis. We show that the deletion of endogenous AAA+ proteases significantly bypasses the need for a dedicated chaperone and identify the mycobacterial ClpXP1P2 complex as the main protease involved in TAC antitoxin degradation. In addition, we show that the ClpXP1P2 degron is located at the extreme C-terminal end of the chaperone addiction (ChAD) region of the antitoxin, demonstrating that ChAD functions as a hub for both chaperone binding and recognition by proteases.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Proteínas de Bactérias/genética , Endopeptidase Clp/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Chaperonas Moleculares/genética , Mycobacterium tuberculosis/genética , Sistemas Toxina-Antitoxina/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Endopeptidase Clp/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Genoma Bacteriano , Chaperonas Moleculares/metabolismo , Mycobacterium tuberculosis/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
11.
FEBS J ; 288(1): 111-126, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32301575

RESUMO

In Mycobacterium tuberculosis (Mtb), the Clp protease degradation pathway, mediated by the modular ClpCP and ClpXP protease complexes, is essential for growth and presents an attractive drug target. Employing a bacterial adenylate cyclase two-hybrid (BACTH) screening approach that we adapted to screen the proteome of an Mtb ORF library, we identify protein interaction partners of the ClpC1 chaperone on a genome-wide level. Our results demonstrate that bipartite type II toxin-antitoxin (TA) systems represent a major substrate class. Out of the 67 type II TA systems known in Mtb, 25 appear as ClpC1 interaction partners in the BACTH screen, including members of the VapBC, MazEF, and ParDE families, as well as a RelBE member that was identified biochemically. We show that antitoxins of the Vap and Rel families are degraded by ClpCP in vitro. We also demonstrate that ClpCP is responsible for mediating the N-end rule pathway, since the adaptor protein ClpS supports ClpC-dependent degradation of an N-end rule model substrate in vitro.


Assuntos
Antitoxinas/genética , Toxinas Bacterianas/genética , Endopeptidase Clp/genética , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/genética , Antitoxinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endopeptidase Clp/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Biblioteca Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Genoma Bacteriano , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Fases de Leitura Aberta , Mapeamento de Interação de Proteínas , Estabilidade Proteica , Proteólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Sistemas Toxina-Antitoxina/genética
12.
Methods Mol Biol ; 2186: 145-155, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32918735

RESUMO

Proteins present a significant challenge for nanopore-based sequence analysis. This is partly due to their stable tertiary structures that must be unfolded for linear translocation, and the absence of regular charge density. To address these challenges, here we describe how ClpXP, an ATP-dependent protein unfoldase, can be harnessed to unfold and processively translocate multi-domain protein substrates through an alpha-hemolysin nanopore sensor. This process results in ionic current patterns that are diagnostic of protein sequence and structure at the single-molecule level.


Assuntos
Endopeptidase Clp/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Bicamadas Lipídicas/metabolismo , Nanoporos , Desdobramento de Proteína , Transporte Proteico
13.
Elife ; 92020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33258771

RESUMO

AAA+ proteases perform regulated protein degradation in all kingdoms of life and consist of a hexameric AAA+ unfoldase/translocase in complex with a self-compartmentalized peptidase. Based on asymmetric features of cryo-EM structures and a sequential hand-over-hand model of substrate translocation, recent publications have proposed that the AAA+ unfoldases ClpA and ClpX rotate with respect to their partner peptidase ClpP to allow function. Here, we test this model by covalently crosslinking ClpA to ClpP to prevent rotation. We find that crosslinked ClpAP complexes unfold, translocate, and degrade protein substrates in vitro, albeit modestly slower than uncrosslinked enzyme controls. Rotation of ClpA with respect to ClpP is therefore not required for ClpAP protease activity, although some flexibility in how the AAA+ ring docks with ClpP may be necessary for optimal function.


Assuntos
Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Reagentes para Ligações Cruzadas , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Proteólise
14.
Elife ; 92020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33319748

RESUMO

The AAA+ protein disaggregase, Hsp104, increases fitness under stress by reversing stress-induced protein aggregation. Natural Hsp104 variants might exist with enhanced, selective activity against neurodegenerative disease substrates. However, natural Hsp104 variation remains largely unexplored. Here, we screened a cross-kingdom collection of Hsp104 homologs in yeast proteotoxicity models. Prokaryotic ClpG reduced TDP-43, FUS, and α-synuclein toxicity, whereas prokaryotic ClpB and hyperactive variants were ineffective. We uncovered therapeutic genetic variation among eukaryotic Hsp104 homologs that specifically antagonized TDP-43 condensation and toxicity in yeast and TDP-43 aggregation in human cells. We also uncovered distinct eukaryotic Hsp104 homologs that selectively antagonized α-synuclein condensation and toxicity in yeast and dopaminergic neurodegeneration in C. elegans. Surprisingly, this therapeutic variation did not manifest as enhanced disaggregase activity, but rather as increased passive inhibition of aggregation of specific substrates. By exploring natural tuning of this passive Hsp104 activity, we elucidated enhanced, substrate-specific agents that counter proteotoxicity underlying neurodegeneration.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Agregação Patológica de Proteínas/patologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , alfa-Sinucleína/metabolismo , Animais , Caenorhabditis elegans , Linhagem Celular , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Escherichia coli , Variação Genética/genética , Células HEK293 , Humanos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Dobramento de Proteína , Deficiências na Proteostase/genética , Deficiências na Proteostase/patologia , Proteína FUS de Ligação a RNA/metabolismo , Saccharomyces cerevisiae
15.
Biochemistry ; 59(44): 4294-4301, 2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-33135889

RESUMO

Cells rely on protein degradation by AAA+ proteases. A well-known example is the hexameric ClpX unfoldase, which captures ATP hydrolysis to feed substrates into the oligomeric ClpP peptidase. Recent studies show that an asymmetric ClpX spiral cycles protein translocation upon ATP hydrolysis. However, how this cycle affects peptide products is less explored in part because ClpP cleavage is thought to be solely defined by sequence constraints. Here, we comprehensively characterize peptides from Caulobacter crescentus ClpXP degradation of three different substrates using high-resolution mass spectrometry and find that cleavage of translocated substrates is driven by factors other than sequence. We report that defined locations in a translocated protein are especially sensitive to cleavage spaced on average every 10-13 residues. These sites are not exclusively controlled by sequence and are independent of bulk changes in catalytic peptidase sites, ATP hydrolysis, or the efficiency of initial recognition. These results fit a model in which processive translocation through ClpX starts at a specific location in a polypeptide and pauses during reset of the ClpX hexamer after a cycle of translocation. Our work suggests that defined peptides, which could be used as signaling molecules, can be generated from a given substrate by a nonspecific peptidase.


Assuntos
Caulobacter crescentus/enzimologia , Endopeptidase Clp/metabolismo , Proteólise , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Endopeptidase Clp/química , Hidrólise , Modelos Moleculares , Conformação Proteica
16.
Commun Biol ; 3(1): 646, 2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-33159171

RESUMO

Over a decade ago Polymerase δ interacting protein of 38 kDa (PDIP38) was proposed to play a role in DNA repair. Since this time, both the physiological function and subcellular location of PDIP38 has remained ambiguous and our present understanding of PDIP38 function has been hampered by a lack of detailed biochemical and structural studies. Here we show, that human PDIP38 is directed to the mitochondrion in a membrane potential dependent manner, where it resides in the matrix compartment, together with its partner protein CLPX. Our structural analysis revealed that PDIP38 is composed of two conserved domains separated by an α/ß linker region. The N-terminal (YccV-like) domain of PDIP38 forms an SH3-like ß-barrel, which interacts specifically with CLPX, via the adaptor docking loop within the N-terminal Zinc binding domain of CLPX. In contrast, the C-terminal (DUF525) domain forms an immunoglobin-like ß-sandwich fold, which contains a highly conserved putative substrate binding pocket. Importantly, PDIP38 modulates the substrate specificity of CLPX and protects CLPX from LONM-mediated degradation, which stabilises the cellular levels of CLPX. Collectively, our findings shed new light on the mechanism and function of mitochondrial PDIP38, demonstrating that PDIP38 is a bona fide adaptor protein for the mitochondrial protease, CLPXP.


Assuntos
Endopeptidase Clp/metabolismo , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Endopeptidase Clp/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Recombinantes
17.
J Am Chem Soc ; 142(49): 20519-20523, 2020 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-33232135

RESUMO

ClpPs are a conserved family of serine proteases that collaborate with ATP-dependent translocases to degrade protein substrates. Drugs targeting these enzymes have attracted interest for the treatment of cancer and bacterial infections due to their critical role in mitochondrial and bacterial proteostasis, respectively. As such, there is significant interest in understanding structure-function relationships in this protein family. ClpPs are known to crystallize in extended, compact, and compressed forms; however, it is unclear what conditions favor the formation of each form and whether they are populated by wild-type enzymes in solution. Here, we use cryo-EM and solution NMR spectroscopy to demonstrate that a pH-dependent conformational switch controls an equilibrium between the active extended and inactive compressed forms of ClpP from the Gram-negative pathogen Neisseria meningitidis. Our findings provide insight into how ClpPs exploit their rugged energy landscapes to enable key conformational changes that regulate their function.


Assuntos
Proteínas de Bactérias/química , Endopeptidase Clp/química , Neisseria meningitidis/enzimologia , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Endopeptidase Clp/metabolismo , Concentração de Íons de Hidrogênio , Conformação Proteica , Termodinâmica
18.
J Bacteriol ; 203(1)2020 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-33020222

RESUMO

Protein degradation is an essential process in all organisms. This process is irreversible and energetically costly; therefore, protein destruction must be tightly controlled. While environmental stresses often lead to upregulation of proteases at the transcriptional level, little is known about posttranslational control of these critical machines. In this study, we show that in Caulobacter crescentus levels of the Lon protease are controlled through proteolysis. Lon turnover requires active Lon and ClpAP proteases. We show that specific determinants dictate Lon stability with a key carboxy-terminal histidine residue driving recognition. Expression of stabilized Lon variants results in toxic levels of protease that deplete normal Lon substrates, such as the replication initiator DnaA, to lethally low levels. Taken together, results of this work demonstrate a feedback mechanism in which ClpAP and Lon collaborate to tune Lon proteolytic capacity for the cell.IMPORTANCE Proteases are essential, but unrestrained activity can also kill cells by degrading essential proteins. The quality-control protease Lon must degrade many misfolded and native substrates. We show that Lon is itself controlled through proteolysis and that bypassing this control results in toxic consequences for the cell.


Assuntos
Caulobacter crescentus/metabolismo , Protease La/metabolismo , Sequência de Aminoácidos , Western Blotting , Caulobacter crescentus/genética , Endopeptidase Clp/genética , Endopeptidase Clp/isolamento & purificação , Endopeptidase Clp/metabolismo , Citometria de Fluxo , Microscopia de Contraste de Fase , Plasmídeos , Protease La/química , Protease La/genética , Protease La/isolamento & purificação , Proteólise
19.
Proc Natl Acad Sci U S A ; 117(41): 25455-25463, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-33020301

RESUMO

ClpA is a hexameric double-ring AAA+ unfoldase/translocase that functions with the ClpP peptidase to degrade proteins that are damaged or unneeded. How the 12 ATPase active sites of ClpA, 6 in the D1 ring and 6 in the D2 ring, work together to fuel ATP-dependent degradation is not understood. We use site-specific cross-linking to engineer ClpA hexamers with alternating ATPase-active and ATPase-inactive modules in the D1 ring, the D2 ring, or both rings to determine if these active sites function together. Our results demonstrate that D2 modules coordinate with D1 modules and ClpP during mechanical work. However, there is no requirement for adjacent modules in either ring to be active for efficient enzyme function. Notably, ClpAP variants with just three alternating active D2 modules are robust protein translocases and function with double the energetic efficiency of ClpAP variants with completely active D2 rings. Although D2 is the more powerful motor, three or six active D1 modules are important for high enzyme processivity, which depends on D1 and D2 acting coordinately. These results challenge sequential models of ATP hydrolysis and coupled mechanical work by ClpAP and provide an engineering strategy that will be useful in testing other aspects of ClpAP mechanism.


Assuntos
Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Trifosfato de Adenosina/metabolismo , Endopeptidase Clp/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica , Variação Genética , Modelos Moleculares , Mutação , Conformação Proteica
20.
Proc Natl Acad Sci U S A ; 117(45): 28005-28013, 2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-33106413

RESUMO

Escherichia coli ClpXP is one of the most thoroughly studied AAA+ proteases, but relatively little is known about the reactions that allow it to bind and then engage specific protein substrates before the adenosine triphosphate (ATP)-fueled mechanical unfolding and translocation steps that lead to processive degradation. Here, we employ a fluorescence-quenching assay to study the binding of ssrA-tagged substrates to ClpXP. Polyphasic stopped-flow association and dissociation kinetics support the existence of at least three distinct substrate-bound complexes. These kinetic data fit well to a model in which ClpXP and substrate form an initial recognition complex followed by an intermediate complex and then, an engaged complex that is competent for substrate unfolding. The initial association and dissociation steps do not require ATP hydrolysis, but subsequent forward and reverse kinetic steps are accelerated by faster ATP hydrolysis. Our results, together with recent cryo-EM structures of ClpXP bound to substrates, support a model in which the ssrA degron initially binds in the top portion of the axial channel of the ClpX hexamer and then is translocated deeper into the channel in steps that eventually pull the native portion of the substrate against the channel opening. Reversible initial substrate binding allows ClpXP to check potential substrates for degrons, potentially increasing specificity. Subsequent substrate engagement steps allow ClpXP to grip a wide variety of sequences to ensure efficient unfolding and translocation of almost any native substrate.


Assuntos
Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica/métodos , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Hidrólise , Cinética , Dobramento de Proteína , Especificidade por Substrato
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